CN117904076A - Novel feruloyl esterase for efficiently degrading polyethylene terephthalate and application thereof - Google Patents
Novel feruloyl esterase for efficiently degrading polyethylene terephthalate and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a novel feruloyl esterase for efficiently degrading polyethylene terephthalate and application thereof. The amino acid sequence of feruloyl esterase WaFae is shown as SEQ ID NO. 1. The characterization result of the feruloyl esterase WaFae constructed by the invention shows that the optimal enzyme activity temperature of the enzyme is 70 ℃ and the optimal enzyme activity pH is 10; at 72 ℃, 90% depolymerization of the low-crystallinity PET powder can be realized within 4 hours, the hydrolytic activity is better than that of the engineering cutinase ICCG, and the high-crystallinity PET plastic with 30% has good degradation activity. The novel feruloyl esterase WaFae has great potential for industrial application.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to novel feruloyl esterase for efficiently degrading polyethylene glycol terephthalate and application thereof.
Background
The rapid development of the plastic industry brings great convenience to daily production and living of people, but due to the large amount of plastic products, the characteristics of low recovery rate, improper treatment mode, difficult natural degradation and the like of plastics, a large amount of plastic garbage is continuously accumulated in natural environment, and serious burden is caused to the global ecological environment. Polyethylene terephthalate (Polyethylene glycol terephthalate, PET) is synthesized from petroleum-derived terephthalic acid and ethylene glycol as materials, and the constituent repeating units are connected through ester bonds to form a linear macromolecular structure. Because of the advantages of low production cost, convenient use and the like, PET becomes one of the polyester materials with the largest use amount, the annual output is up to 6000 ten thousand tons, and more than half of the global synthetic fibers, clothing textiles and plastic bottles are made of PET. The waste PET plastic is mainly recovered physically, but the physical degradation recovery problem exists, and the recovery cycle times are small; however, the chemical recovery introduces impurities, which results in degradation of the quality of the refolded PET, so that development of an economic, green and cyclic recovery method is needed, the power-assisted plastic is recycled, the ecological environment is protected, and natural resources are saved.
Microbial enzyme-mediated degradation treatment of PET is the most promising and environmentally friendly strategy for achieving sustainable development of PET plastics. A number of PET hydrolases have been discovered which degrade PET to ethylene terephthalate (BHET), monoethylene terephthalate (MHET) or terephthalic acid (TPA) for efficient recycling. It has been reported that cutinases, lipases and esterase family members possess PET hydrolytic activity, but most hydrolases exhibit limited PET degradation capacity due to low substrate specificity and poor turnover efficiency. The PET molecules are regularly arranged, the vitrification temperature is 67-80 ℃, and the hydrolase which is stable at high temperature is the key for efficiently degrading PET. In order to realize green and efficient recycling of PET plastics, it is highly desirable to search for PET hydrolytic enzymes which are stable at high temperatures and have high degradation efficiency. Because of the specificity of the living environment, most of the thermophilic microorganisms have good high-temperature tolerance and are ideal sources for digging high-temperature stable PET hydrolase. Through metagenome excavation, a novel feruloyl esterase WaFae is discovered from hot spring archaea Candidatus Wolframiiraptor allenii, the optimal enzyme reaction temperature is 70 ℃, PET plastic can be efficiently degraded, and the good industrial application potential is shown.
Disclosure of Invention
The first object of the invention is to provide a novel feruloyl esterase WaFae, the amino acid sequence of which is shown as SEQ ID NO. 1.
The second object of the present invention is to provide a coding gene encoding the above feruloyl esterase WaFae.
Preferably, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.
A third object of the present invention is to provide a biological material containing the above-mentioned coding gene.
Preferably, the biological material is a recombinant vector, an expression cassette, a host cell or a recombinant engineering bacterium.
Preferably, the recombinant vector is pET-21a.
Preferably, the host bacterium of the recombinant engineering bacterium is escherichia coli.
Preferably, the E.coli is BL21 (DE 3).
It is a fourth object of the present invention to provide a product comprising the above feruloyl esterase WaFae or a biological material.
It is a fifth object of the present invention to provide the use of the above feruloyl esterase WaFae, a biological material or a product in the degradation of PET or a substance containing PET plastic.
It is a sixth object of the present invention to provide a process for degrading PET or PET-containing plastic materials by reacting PET with feruloyl esterase WaFae, a biological material or a product as described above.
Preferably, the ratio of feruloyl esterase WaFae to PET substrate is 2mg (enzyme)/g (PET).
According to the invention, through a metagenome excavation mode, feruloyl esterase WaFae is found to show high-efficiency PET degradation activity, which is superior to the catalytic activity of the currently reported PET hydrolase ICCG, and has better industrial application potential in the aspects of reducing production cost, improving production efficiency and the like.
Drawings
FIG. 1 is a comparison of WaFae (purple) and PET hydrolase 204 (powder) predicted structures.
FIG. 2 is a comparison of catalytic activity at WaFae different temperatures and pH (A is catalytic activity at different temperatures and B is catalytic activity at different pH).
FIG. 3 is a comparison of the degradation effects of WaFae and ICCG on PET materials of different crystallinity (A is a comparison of the catalytic activity of PET films, B is a comparison of the catalytic activity of PET powders of different crystallinity, where 1 is 10% crystallinity and 2 is 30% crystallinity).
Fig. 4 is a comparison of deagglomeration efficiency of WaFae and ICCG for low crystallinity PET powders at high density.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
The following examples are not specific for molecular biological assays, and are all made with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) J.Sam Brookfield, or according to the kit and product instructions.
The following definitions are employed in the present invention:
1. Nomenclature of amino acids and DNA nucleotide sequences
Using the accepted IUPAC nomenclature for amino acid residues, the code is in three letter code. The DNA nucleotide sequence uses accepted IUPAC nomenclature.
Example 1: excavation and structural analysis of feruloyl esterase sequences
The cutinase ScCut previously discovered in the subject group shows high PET degradation efficiency and strong affinity, and the structure is analyzed to find that the enzyme is provided with a special 'cover' domain which is the basis of high activity. In order to mine efficient PET hydrolase, a protein structure comparison based on Alpha fold 2 prediction is carried out by taking cutinase ScCut (GenBank: UPK 75129.1) as a template, and the archaea source hydrolase in a hot spring sample metagenome is found to be named WaFae temporarily, and has a unique cover domain and a binding pocket and has a certain structural similarity with ScCut.
Through sequence analysis, waFae belongs to feruloyl esterase, is an alpha/beta hydrolase superfamily, contains a catalytic triplet Ser-His-Asp and G-X-S-X-G conserved structure, has 2 disulfide bonds (C3-C58, C83-C179), and has an amino acid sequence shown in SEQ ID NO. 1. The maximum similarity of WaFae gene sequences to the reported PET hydrolase 204 was found in the NCBI database to be 68% (GenBank: WP-187147021.1,Nature Communications,2022, 13: 7850), whereas WaFae had a longer loop, possibly favoring substrate binding (FIG. 1).
The amino acid sequence of feruloyl esterase WaFae is shown as SEQ ID NO.1, specifically:
MSCREEFVKIPSAGFELAGVLHLPIGTTKPKPVLFLHGFTGNKVEAGRMYTDMARVLCAAGYASLRFDFRCHGDSPLPFEEYCISYAIEDARNAANFLKNLTSIDDLRFAIIGLSMGGGVAVNLAANRDDIAALVLLSPALDWPELTSILSQWKVEGDYVYMGPPGSPSPRLYRMKINCATEMMKFSVMGLAELIKAPTLIIHAIDDVVIPISQAKRFFERLKVEKKFIEIEHGGHVFEDYDTRRRIEKEILDWLKQYF.
Example 2: obtaining feruloyl esterase-producing Strain
The feruloyl esterase WaFae sequence is subjected to codon optimization and then sent to Tianyihuo (Guangzhou) biological company for synthesis, and the gene sequence is shown as SEQ ID NO. 2. After gene synthesis, 6 histidine encoding genes were added to the C-terminus of the sequence and ligated between EcoRI-HindIII cloning sites of the pET-21a plasmid. Adding 4 mu L of constructed plasmid pET-21a-WaFae into 100 mu L of E.coli BL21 (DE 3) competent cells, carrying out ice bath for 30min, then carrying out heat shock at 42 ℃ for 90s, carrying out ice bath reaction for 2min, adding 500 mu L of LB culture medium (without antibiotics) into a centrifuge tube, culturing for 1h at 37 ℃ and 180r/min, coating the E.coli cells on an LB culture dish containing ampicillin (100 mu g/mL), culturing for 12h at 37 ℃, picking up monoclonal to carry out sequencing verification, and obtaining the genetically engineered bacterium BL21 (DE 3)/WaFae containing ferulic acid esterase after verification success.
The nucleotide sequence of the feruloyl esterase WaFae coding gene is shown as SEQ ID NO.2, and specifically comprises the following steps:
GAATTCATGAGCTGTCGCGAAGAATTTGTTAAGATTCCGAGCGCGGGGTTTGAATTAGCGGGGGTTCTGCACCTGCCGATTGGTACCACGAAACCGAAACCGGTTCTGTTTTTACACGGTTTTACCGGTAATAAAGTTGAAGCCGGGCGCATGTATACCGATATGGCCCGTGTTTTATGTGCAGCAGGTTATGCAAGCCTGAGATTTGATTTTCGTTGTCATGGTGATTCTCCTCTGCCGTTTGAAGAGTATTGTATTAGTTATGCAATCGAGGATGCACGGAATGCCGCAAATTTTTTAAAGAATCTGACCTCAATCGACGATTTACGTTTTGCCATTATTGGTTTAAGCATGGGTGGTGGGGTTGCTGTTAACCTGGCGGCAAATAGAGATGATATAGCCGCGCTGGTGCTGCTGAGCCCGGCTTTAGATTGGCCGGAACTGACATCGATTCTGAGCCAGTGGAAAGTTGAAGGTGATTATGTGTATATGGGTCCTCCGGGGAGCCCGAGCCCGAGATTATATCGGATGAAAATAAATTGTGCCACGGAAATGATGAAATTTAGCGTGATGGGATTAGCGGAACTGATTAAGGCACCGACGCTGATTATTCACGCAATAGATGATGTTGTTATCCCGATTAGCCAGGCAAAACGCTTTTTTGAACGGTTAAAGGTGGAGAAAAAGTTTATTGAGATCGAACATGGTGGACATGTCTTCGAAGACTATGATACACGCAGACGGATTGAGAAAGAAATTCTGGATTGGCTGAAACAGTATTTTAAGCTT.( Underlined are cleavage sites).
Example 3: preparation of novel feruloyl esterase WaFae protein
Inoculating genetically engineered bacterium BL21 (DE 3)/WaFae into LB liquid culture medium containing ampicillin (100 mug/mL) according to the inoculum size of 2% of volume ratio, and shake culturing at 37 ℃ and 180rpm/min until OD600 is 0.6-0.8; IPTG was then added at a final concentration of 1mM and cultured with shaking at 16℃for 20 hours. The cells were collected by centrifugation at 8000r/min at 4℃for 10min, washed 3 times with phosphate buffer (500 mM Na 2HPO4, 50mM NaCl, pH 8.0), resuspended and sonicated, and the precipitate was removed by centrifugation at 10000r/min at 4℃to give a supernatant as a crude enzyme solution. The crude enzyme was purified by Ni 2+ affinity chromatography column, eluted with imidazole gradient, the eluate was collected, then replaced with 500mM phosphate buffer pH8.0 using a 10kDa ultrafiltration tube, and concentrated. The purity of the protein was checked by SDS-PAGE, and the concentration of the concentrated protein was checked by an ultramicro protein detector. Thereby obtaining feruloyl esterase WaFae.
Example 4: analysis of PET plastics degradation efficiency by feruloyl esterase WaFae
The feruloyl esterase WaFae is used for carrying out degradation experiments on PET films and powder according to the following steps:
PET film degradation experimental procedure: cutting a PET film into a circular sheet with the diameter of 8mm, adding 0.5mL of 1M phosphate solution (pH 8.5) into a 2mL EP tube, putting 1 piece of PET plastic film, and then adding 30 mug of purified ferulic acid esterase, wherein the material ratio is 2mg (enzyme)/g (PET); the control group was prepared by adding 0.5mL of a 1M phosphate solution (pH 8.5) and placing 1 piece of PET plastic film. Three replicates were set for each treatment, and the EP tube was placed in a water bath to detect PET degradation products. ICCG enzyme was used as a control. The reaction was terminated by boiling at high temperature and the product was detected.
PET powder degradation experimental procedure: 15mg (crystallinity 10.7%) of PET powder was weighed into a 2mL EP tube, followed by the addition of 0.5mL of 1M phosphate solution (pH 8.5) and 30. Mu.g of purified feruloyl esterase in a material ratio of 2mg (enzyme)/g (PET); the control group was added with 0.5mL of 1M phosphate solution (pH 8.5) and 15mg of PET powder. Three replicates were set for each treatment, and the EP tube was placed in a water bath to detect PET degradation products. The reaction was stopped by boiling at high temperature using ICCG enzyme as a control, and the product was detected.
3. Determination of the optimal reaction temperature and pH of feruloyl esterase WaFae: PET film is selected as a substrate, the prepared reaction systems are respectively placed at 40, 50, 60, 65, 70 and 80 ℃ for reaction for 4 hours, and then the amount of reaction products is detected to determine the optimal reaction temperature of feruloyl esterase WaFae. Preparing 1M phosphate solution (pH 6-11), selecting PET film as substrate, placing the prepared reaction systems at 70 ℃ for reaction for 4 hours respectively, detecting the amount of reaction products, and determining the optimal pH.
Detection flow of PET degradation products: 20. Mu.L of the degradation solution was diluted 50-fold with ultrapure water and filtered through a 0.22 μm aqueous filter membrane for use. The composition of TPA, MHET and BHET was detected using agilent 1260 high performance liquid chromatography. The column was Zorbax SB-C18ODS (4.6X105 mm,5 μm) and the detection wavelength was 240nm. Mobile phase: a is deionized water containing 0.1% (v/v) formic acid; b is methanol containing 0.1% (v/v) formic acid. Gradient elution conditions: 0-5min 10% B;5-20min of 10% -100% B solution; the flow rate was 1.0mL/min and the column temperature was maintained at 30 ℃.
To determine the optimal reaction temperature of feruloyl esterase WaFae, the added solutions were reacted at 40, 50, 60, 65, 70, 80℃for 4 hours, respectively, and then the amount of the reaction product was measured. The feruloyl esterase WaFae was determined to exhibit an optimal catalytic activity at 70℃and an increase in activity with increasing temperature between 40 and 70 ℃. The enzyme activity decreased dramatically at 80 ℃ (figure 2A). Feruloyl esterase WaFae showed the best activity at pH 10, with catalytic activity greater than 60% in the pH range 8-11 (FIG. 2B).
When a low crystallinity PET film was used as the reaction substrate, waFae had a product release of 3.6 times ICCG at 65 ℃ and 3.4 times ICCG at 72 ℃ (fig. 3A), with significantly better hydrolysis efficiency than the currently reported engineered PET hydrolase ICCG. When powders with different crystallinity (10% and 30%) are used as reaction substrates, the degradation efficiency of WaFae under the reaction condition of 72 ℃ is higher than ICCG. It is worth mentioning that WaFae has significantly better degradation efficiency for 30% crystallinity PET plastics than ICCG (fig. 3B).
Under the conditions of high concentration substrate (60 g/L) 72 ℃ and pH 8.5, waFae can realize 90% depolymerization of PET powder in 4h and 95% depolymerization of PET powder in 8h (figure 4), which is superior to the activity of the currently reported PET hydrolase ICCG, and can completely meet the recycling requirement of industrial waste PET plastics.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (10)
1. A novel feruloyl esterase is characterized in that the amino acid sequence of the novel feruloyl esterase is shown as SEQ ID NO. 1.
2. A coding gene encoding the feruloyl esterase of claim 1.
3. The coding gene according to claim 2, wherein the nucleotide sequence is shown in SEQ ID NO. 2.
4. A biological material comprising the coding gene of claim 2.
5. The biomaterial of claim 4, wherein the biomaterial is a recombinant vector, an expression cassette, a host cell, or a recombinant engineering bacterium.
6. The biomaterial of claim 4, wherein the recombinant vector is pET-21a.
7. The biomaterial according to claim 4, wherein the host bacterium of the recombinant engineering bacterium is Escherichia coli.
8. A product comprising the feruloyl esterase of claim 1 or the biological material of claim 4.
9. Use of the feruloyl esterase of claim 1, the biomaterial of claim 4 or the product of claim 8 for degradation of polyethylene terephthalate or a substance containing polyethylene terephthalate plastic.
10. A process for degrading polyethylene terephthalate or a material comprising polyethylene terephthalate plastic, characterized in that the feruloyl esterase according to claim 1, the biomaterial according to claim 4 or the product according to claim 8 is reacted with polyethylene terephthalate.
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